The tumor suppressor Kruppel-like factor 6 is a novel aryl hydrocarbon receptor DNA binding partner.

The aryl hydrocarbon receptor (AhR) is a ligand-mediated basic helix-loop-helix transcription factor of the Per/Arnt/Sim family that regulates adaptive and toxic responses to a variety of chemical pollutants, including polycyclic aromatic hydrocarbons and halogenated aromatic hydrocarbons, most notably 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Ligand activation leads to AhR nuclear translocation and binding to a xenobiotic response element (XRE) in association with the Arnt to regulate gene expression. Several recent genome-wide transcriptional studies identified numerous AhR target genes that lack the canonical XRE recognition site in the promoter regions. Characterization of one such target gene, the plasminogen activator inhibitor 1, identified a novel nonconsensus XRE (NC-XRE) that confers TCDD responsiveness independently of the Arnt protein. Studies reported here show that the NC-XRE is a recognition site for the AhR and a new binding partner, the Kruppel-like factor (KLF) family member KLF6. In vivo chromatin immunoprecipitations and in vitro DNA binding studies demonstrate that the AhR and KLF6 proteins form an obligatory heterodimer necessary for NC-XRE binding. Mutational analyses show that the protein-protein interactions involve the AhR C terminus and KLF6 N terminus, respectively. Moreover, NC-XRE binding depends on the 5' basic region in KLF6 rather than the previously characterized zinc finger DNA binding domain. Collectively, the results unmask a novel AhR signaling mechanism distinct from the canonical XRE-driven process that will enrich our future understanding of AhR biology.

The activated aryl hydrocarbon receptor synergizes mitogen-induced murine liver hyperplasia.

Mechanisms of hepatocyte proliferation triggered by tissue loss are distinguishable from those that promote proliferation in the intact liver in response to mitogens. Previous studies demonstrate that exogenous activation of the aryl hydrocarbon receptor (AhR), a soluble ligand-activated transcription factor in the basic helix-loop-helix family of proteins, suppresses compensatory liver regeneration elicited by surgical partial hepatectomy. The goal of the present study was to determine how AhR activation modulates hepatocyte cell cycle progression in the intact liver following treatment with the hepatomitogen, 1,4-bis[2-(3,5-dichloropyridyloxy)] benzene (TCPOBOP). Mice were pretreated with the exogenous AhR agonist 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) 24h prior to treatment with TCPOBOP (3 mg/kg).). In contrast to the suppressive effects of AhR activation observed during compensatory regeneration, TCDD pretreatment resulted in a 30-50% increase in hepatocyte proliferation in the intact liver of TCPOBOP-treated mice. Although pretreatment with TCDD suppressed CDK2 kinase activity and increased the association of CDK2 with negative regulatory proteins p21Cip1 and p27Kip1, a corresponding increase in CDK4/cyclin D1 association and CDK4 activity which culminated in enhanced phosphorylation of retinoblastoma protein, consistent with the increased proliferative response. These findings are in stark contrast to previous observations that the activated AhR can suppress hepatocyte proliferation in vivo and reveal a new complexity to AhR-mediated cell cycle control.

Hypoxia exposure induces the emergence of fibroblasts lacking replication repressor signals of PKCzeta in the pulmonary artery adventitia.

AIMS: Cultured fibroblasts of hypoxia-stimulated remodelled pulmonary artery (PA) adventitia proliferate at a greater rate compared with those of normal adventitia. Since protein kinase C (PKC) zeta is a replication repressor of normal adventitial fibroblasts, we hypothesized that loss of the repressor activity of PKCzeta might contribute to increased rate of proliferation in adventitial cells of remodelled PA. METHODS AND RESULTS: Isolated PA adventitial fibroblasts of neonatal control (Fib-C) and chronic hypoxia-exposed (Fib-H) calves were used to test our hypothesis. For evaluation of the role of PKCzeta in hypoxia-induced vascular adventitial remodelling, expression and activation of PKCzeta were also examined in lung sections of Fib-C and Fib-H animals by immunoperoxidase staining. Although constitutively active PKCzeta expression attenuated DNA synthesis in Fib-C, it stimulated proliferation in Fib-H. PKCzeta-specific myristoylated pseudosubstrate peptide inhibitor (PKCzeta-PI) induced replication in Fib-C, whereas the inhibitor blocked DNA synthesis in Fib-H. Hypoxia stimulated PKCzeta as well as MAP kinase kinase (MEK)1/2 and extracellular signal-regulated kinase (ERK)1/2 phosphorylation in Fib-H cells. However, ERK1/2 activation was mediated by both MEK1/2-dependent and MEK1/2-independent PKCzeta-regulated mechanisms in hypoxia-exposed Fib-H. PKCzeta was selectively activated in the adventitial cells of the remodelled vascular wall, as demonstrated by strong immunoreactivity against the anti-phosphoPKCzeta antibody in the Fib-H lung sections. CONCLUSION: PKCzeta acts as a replication repressor in Fib-C cells; however, the same isozyme mediates Fib-H proliferation. Thus, chronic exposure to hypoxia leads to the emergence of cells lacking anti-replication activity of PKCzeta in the PA adventitia.

Optimization and validation of an ischemic wound model.

Localized tissue ischemia is a key factor in the development and poor prognosis of chronic wounds. Currently, there are no standardized animal models that provide sufficient tissue to evaluate the effect of modalities that may induce angiogenesis, and in vitro models of angiogenesis do not mimic the complexity of the ischemic wound bed. Therefore, we set out to develop a reproducible ischemic model for use in wound-healing studies. Male Sprague-Dawley rats underwent creation of dorsal bipedicle skin flaps with centrally located excisional wounds. Oxygen tension, wound-breaking strength, wound area, lactate, and wound vascular endothelial growth factor (VEGF) were compared in flaps measuring 2.5 and 2.0 x 11 cm with and without an underlying silicone sheet. We found that the center of the 2.0 cm flap with silicone remains in the critically ischemic range up to 14 days without tissue necrosis (33+/-4 vs. 49+/-6 mmHg in controls). Wound healing and breaking strength were significantly impaired and tissue lactate from the center of this flap was 2.9 times greater than tissue from either nonischemic controls and 2.5 cm flap (0.23+/-0.05 mg/dL/mg sample vs. 0.09+/-0.02 and 0.08+/-0.02, respectively). Vascular endothelial growth factor was 2 times greater than the nonischemic control. This ischemic wound model is relatively inexpensive, easy to perform, reproducible, and reliable. The excisional wounds provide sufficient tissue for biochemical and histologic analysis, and are amenable to the evaluation of topical and systemic therapies that may induce angiogenesis or improve wound healing.

Zeamatin, clotrimazole and nikkomycin Z in therapy of a Candida vaginitis model.

OBJECTIVE: To study the interaction of antifungal drugs in topical therapy. MATERIALS AND METHODS: Local therapy of Candida vaginitis with drugs alone and in combination was examined in a murine model. Zeamatin, a natural plant-derived antifungal protein, was tested alone and in combination with an azole, clotrimazole or nikkomycin Z, a chitin synthase inhibitor. RESULTS: Whereas alone, zeamatin was ineffective, nikkomycin Z was effective only when dosed multiple times per day, and clotrimazole efficacy was variable when administered in experimental vehicles (unlike the complex and undefined commercial preparation), zeamatin enhanced the efficacy of either of the other two drugs when they were given in combination. CONCLUSION: Drug interactions between novel drugs with unique mechanisms of action should be explored further, and may lead to more potent regimens.